Kari T. Steffen

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

Abstract Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

Degradation of Humic Acids by the Litter-Decomposing Basidiomycete Collybia dryophila

ABSTRACT The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic 14C-labeled humic acid (14C-HA) prepared from [U-14C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn2+ (200 μM), leading to 75% conversion of LHA and 50% mineralization of 14C-HA (compared to 60% and 20%, respectively, in the absence of Mn2+). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn2+-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.

Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase.

ABSTRACT The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn2+) supplied at a concentration of 200 μM stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn2+ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn2+. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter−1 was converted in an in vitro reaction solution containing 1 U of MnP ml−1 within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter−1) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).

Purification and characterization of manganese peroxidases from the litter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla

Abstract Extracellular manganese peroxidase (MnP) was purified from liquid cultures of the litter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla. Both fungi produced MnP increasingly in response to Mn 2+ in the medium. A. praecox secreted two MnP isoforms with similar isoelectric points (p I ) of 6.3–7.0 and a molecular weight (MW) of 42 kDa. MnP activity was not observed in Mn 2+ -free cultures of A. praecox. In Mn 2+ -supplemented cultures, S. coronilla produced at least two MnPs, of which the main isoform MnP1 has a p I of 6.3–7.1 and a MW of 41 kDa. In addition, S. coronilla possesses a partly constitutive MnP (MnP2) which was also detectable in Mn 2+ -free cultures, although its amount was considerably lower. MnP2 showed two distinct bands with acidic p I s of 3.5 and 3.7 in the IEF gel and has a MW of 41 kDa. There are indications for the existence of a third, likewise Mn 2+ -inducible enzyme (MnP3), that could not be separated from MnP2 but formed an additional band in eletrophoretic analyses (p I 5.1, MW 43 kDa).

Potential of cometabolic transformation of polysaccharides and lignin in lignocellulose by soil Actinobacteria

While it is known that several Actinobacteria produce enzymes that decompose polysaccharides or phenolic compounds in dead plant biomass, the occurrence of these traits in the environment remains largely unclear. The aim of this work was to screen isolated actinobacterial strains to explore their ability to produce extracellular enzymes that participate in the degradation of polysaccharides and their ability to cometabolically transform phenolic compounds of various complexities. Actinobacterial strains were isolated from meadow and forest soils and screened for their ability to grow on lignocellulose. The potential to transform 14C-labelled phenolic substrates (dehydrogenation polymer (DHP), lignin and catechol) and to produce a range of extracellular, hydrolytic enzymes was investigated in three strains of Streptomyces spp. that possessed high lignocellulose degrading activity. Isolated strains showed high variation in their ability to produce cellulose- and hemicellulose-degrading enzymes and were able to mineralise up to 1.1% and to solubilise up to 4% of poplar lignin and to mineralise up to 11.4% and to solubilise up to 64% of catechol, while only minimal mineralisation of DHP was observed. The results confirm the potential importance of Actinobacteria in lignocellulose degradation, although it is likely that the decomposition of biopolymers is limited to strains that represent only a minor portion of the entire community, while the range of simple, carbon-containing compounds that serve as sources for actinobacterial growth is relatively wide.

Scots pine (Pinus sylvestris) bark composition and degradation by fungi: potential substrate for bioremediation.

The composition of Scots pine bark, its degradation, and the production of hydrolytic and ligninolytic enzymes were evaluated during 90 days of incubation with Phanerochaete velutina and Stropharia rugosoannulata. The aim was to evaluate if pine bark can be a suitable fungal substrate for bioremediation applications. The original pine bark contained 45% lignin, 25% cellulose, and 15% hemicellulose. Resin acids were the most predominant lipophilic extractives, followed by sitosterol and unsaturated fatty acids, such as linoleic and oleic acids. Both fungi degraded all main components of bark, specially cellulose (79% loss by P. velutina). During cultivation on pine bark, fungi also degraded sitosterol, produced malic acid, and oxidated unsaturated fatty acids. The most predominant enzymes produced by both fungi were cellulase and manganese peroxidase. The results indicate that Scots pine bark supports enzyme production and provides nutrients to fungi, thus pine bark may be suitable fungal substrate for bioremediation.

Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus

The soil mold Paecilomyces inflatus is capable of modifying and partially mineralizing synthetic and natural humic acids (HAs) in compost environments. HA degradation studies using a synthetic HA (14C-HA) in autoclaved compost microcosms showed that, after 12 weeks of cultivation, P. inflatus mineralized approximately 5% of the 14C-labeled HA to14CO2, while 6% of the 14C-HA was converted into 14C-labeled water-soluble fragments (fulvic-acid-like fraction). About 40% was still present as NaOH-soluble HA representing unmodified or only slightly modified humic material (compared with 60% in the controls). Modification of natural HAs extracted from compost was followed by their partial decolorization (30%) in liquid cultures of P. inflatus. Bleaching of the medium was accompanied by moderate changes in the molecular mass distribution of both the HA and fulvic-acid fractions, which were analyzed with high-performance size exclusion chromatography. HA modification was most pronounced during the primary growth phase of the fungus and was associated with increased laccase activity.

Fate of bisphenol A during treatment with the litter-decomposing fungi Stropharia rugosoannulata and Stropharia coronilla

Bisphenol A is an endocrine disrupting compound, which is ubiquitous in the environment due to its wide use in plastic and resin production. Seven day old cultures of the litter-decomposing fungus Stropharia coronilla removed the estrogenic activity of bisphenol A (BPA) rapidly and enduringly. Treatment of BPA with purified neutral manganese peroxidase (MnP) from this fungus also resulted in 100% reduction of estrogenic activity, as analyzed using a bioluminescent yeast assay, and in the formation of polymeric compounds. In cultures of Stropharia rugosoannulata, estrogenic activity also quickly disappeared but temporarily re-emerged in the further course of cultivation. LC-MS analysis of the extracted estrogenic culture liquid revealed [M-H](-) ions with m/z values of 219 and 235. We hypothesize that these compounds are ring fission products of BPA, which still exhibit one intact hydroxyphenyl group to interact with estrogen receptors displayed by the yeast.

Bioluminescent Yeast Assay for Detection of Organotin Compounds

Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.

Fungal enzyme production and biodegradation of polychlorinated dibenzo-p-dioxins and dibenzofurans in contaminated sawmill soil

The current treatment method for PCDD/F-contaminated soil, which fulfils the requirements for POP soils, is incineration at high temperature. In this study, we investigated if bioaugmentation with fungal inoculum or treatment with manganese peroxidase (MnP) enzyme preparation could be used instead. The main source of PCDD/F contamination in Finland has been the national production and use of a chlorophenol containing wood preservative, which contained PCDD/Fs as impurities. Therefore, historically contaminated soils from three sawmill sites were used in the experiments. In bioaugmentation experiments with living fungal mycelia, enzyme production, CO2 production and degradation of chlorinated dioxins were measured. When cell free MnP preparation was added to the soil, it was likewise important to follow how enzyme activity was maintained in the soil. As a result of this study, we showed that fungi were able to efficiently degrade PCDD/F, but surprisingly the addition of MnP preparation did not have any effect to the PCDD/F concentration. However, substantial amounts of MnP activity were found in the soil still after 10d of incubation. Treatment with either Stropharia rugosoannulata or Phanerochaete velutina resulted in 62-64% decrease in WHO-TEQ value in 3months. One critical factor for efficient biodegradation was strong growth of fungal mycelia in non-sterile contaminated soil.